During the isolation and characterization of cDNA clones for ADP-ribosylation factors, about 20 kDa GTP-binding proteins, a human brain cDNA containing a partial open reading frame encoding 48 amino acids, of which 10 were basic, was isolated. This peptide was unusual based on its predicted pI of 11.7. Oligonucleotide probes specific for this sequence hybridized with two abundant mRNAs in HL-60 cells, one of which appeared to comigrate with an ARF3 mRNA. Because of the unusual nature of this peptide as well as the size and abundance of the mRNAs, studies were performed to identify and investigate the relationship between these mRNAs. The cDNA fragment was used to isolate overlapping clones from dibutyryl cAMP-differentiated HL-60 and bovine retinal cDNA libraries. A putative open reading frame was identified that encodes a 23.6 kDa protein (p23, 203 amino acids) with a predicted pI of 11.6. The deduced amino acid sequence is highly conserved between human and bovine, exhibiting 97% identity; they are 99% identical if conservative substitutions are included. The human and bovine nucleotide sequences exhibit 91% identity across the coding region. Clones from both species contain a poly-adenylation signal (AATAAA) 4 nucleotides downstream (3') from the termination codon as well as a poly(A)+ tail. Other clones, however, extended beyond the point of poly(A)+ addition. In one bovine clone, the 3'-untranslated region extended 442 nucleotides beyond the polyadenylation signal to end in a poly(A)+ tail; a second polyadenylation signal was found 21 nucleotides before the poly (A)+ tail. Hybridization of poly(A)+ RNA from various bovine tissues and brains of several mammalian species with cDNA and oligonucleotides specific for the coding region identified two abundant mRNA species of 1.2- and 0.8-kb. Oligonucleotide probes specific for the extended 3'-untranslated region hybridized with the 1.2-kb, but not the 0.8-kb, mRNA. Both mRNAs were observed in all mammalian tissues and species. These studies indicate that a ubiquitous, basic 23.6 kDa protein is highly conserved among mammals and is encoded by two mRNA species that appear to differ in the site of polyadenylation.